Characterization of Two Novel Bacillus Thuringiensis Cry8 Toxins

toxins

Article Characterization of Two Novel Bacillus thuringiensis Cry8 Toxins Reveal Diﬀerential Speciﬁcity of Protoxins or Activated Toxins against Chrysomeloidea Coleopteran Superfamily

Changlong Shu 1, Guixin Yan 1, Shizhi Huang 1, Yongxin Geng 1, Mario Soberón 2 , Alejandra Bravo 2 , Lili Geng 1 and Jie Zhang 1,*

1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; [email protected] (C.S.); [email protected] (G.Y.); [email protected] (S.H.); [email protected] (Y.G.); [email protected] (L.G.) 2 Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca 62250, Mexico; [email protected] (M.S.); [email protected] (A.B.) * Correspondence: [email protected]; Tel.: +86-10-62812642

Received: 14 September 2020; Accepted: 2 October 2020; Published: 5 October 2020

Abstract: Scarabaeoidea and Chrysomeloidea insects are agriculture-destructive coleopteran pests. Few eﬀective Bacillus thuringiensis (Bt) insecticidal proteins against these species have been described. Bt isolate BtSU4 was found to be active against coleopteran insects. Genome sequencing revealed two new cry8 genes in BtSU4, designated as cry8Ha1 and cry8Ia1. Both genes expressed a 135 kDa protoxin forming irregular shape crystals. Bioassays performed with Cry8Ha1 protoxin showed that it was toxic to both larvae and adult stages of Holotrichia parallela, also to Holotrichia oblita adults and to Anoplophora glabripennis larvae, but was not toxic to larval stages of H. oblita or Colaphellus bowringi. The Cry8Ia1 protoxin only showed toxicity against H. parallela larvae. After activation with chymotrypsin, the Cry8Ha1 activated toxin lost its insecticidal activity against H. oblita adults and reduced its activity on H. parallela adults, but gained toxicity against C. bowringi larvae, a Chrysomeloidea insect pest that feeds on crucifer crops. The chymotrypsin activated Cry8Ia1 toxin did not show toxicity to any one of these insects. These data show that Cry8Ha1 and Cry8Ia1 protoxin and activated toxin proteins have diﬀerential toxicity to diverse coleopteran species, and that protoxin is a more robust protein for the control of coleopteran insects.

Keywords: Bacillus thuringiensis; cry8 genes; Coleopteran; insecticidal activity; Cry protoxin; dual mode of action

Key Contribution: The study shows that Cry8Ha1 protein has a broad insecticidal spectrum against Chrysomeloidea and Scarabaeoidea pests.

1. Introduction The Coleoptera (beetles) insect order contains more described species than any other animal group, with over 380,000 named species [1,2], comprising almost 40% of described insects and nearly 30% of all animal species. Among these beetles, the species from Scarabaeoidea and Chrysomeloidae superfamilies are considered as the most destructive coleopteran pests, which cause severe damages in agriculture, horticulture, and forestry. The insecticide formulations based on Bacillus thuringiensis (Bt) insecticidal proteins, as well as transgenic plants expressing Bt insecticidal proteins are applied as

Toxins 2020, 12, 642; doi:10.3390/toxins12100642 www.mdpi.com/journal/toxins Toxins 2020, 12, 642 2 of 11 sustainable control methods for some species of these coleopteran pests [3,4]. However, comparing to the beetle’s diversity, the currently discovered variability of Bt insecticidal proteins effective against coleopteran insects is scarce. Currently, only few members in Cry1, Cry3, Cry6 (now APP6), Cry7 Cry8, Cry22 (now XPP22), Cry23/Cry37 (now MPP23/XPP37), Cry34/Cry35 (now GPP34/TPP35), Cry55 (now XPP55), Cyt1, Cyt2, Sip (now MPP5), and Vip1/Vip2 subgroups of Bt protein families were reported to be toxic against certain coleopteran insects [5,6]. Hence, screening of Bt strains and identification of novel insecticidal proteins active against coleopteran insects, especially proteins that display a broad insecticidal spectrum against Chrysomeloidea and Scarabaeoidea is still needed for the effective control of these pests. Bt insecticidal crystal proteins (ICPs) are synthetized as protoxins which, once ingested by the insect, are required to be solubilized, and proteolytically activated in the insect midgut [7,8]. The activated toxin binds to membrane receptors and lyses midgut epithelial cells by forming pores that cause cell swelling. Some of the Cry toxins with potential activity are only toxic after in vitro solubilization and activation with commercial proteases. For example, the toxicity of Cry7Aa towards coleopteran insect larvae was revealed only after an in vitro solubilization and activation with proteases [9]. For this reason, it is recommended to perform bioassays with both protoxins and activated toxins to detect insecticidal activity of a new Bt ICPs, especially against coleopteran larvae. In this report, we screened a new Bt strain, BtSU4, with insecticidal activity against Holotrichia parallela and we cloned two novel cry8-type genes from this strain. Furthermore, the expression, solubilization, activation and toxicity assays of the two novel ICPs indicated that the new Cry8Ha1 protein has a broad insecticidal spectrum important for the control of several important coleopteran pests.

2. Results

2.1. Cloning and Sequence Analysis of cry8Ha1 and cry8Ia1 from BtSU4 After BtSU4 genome sequencing and insecticidal gene annotation, two genes coding for proteins with similarity to Cry8 family were discovered. The two Cry8 proteins consisted of 1199 and 1194 amino acids, respectively. The two novel proteins were subsequently named as Cry8Ha1 and Cry8Ia1 (https://www.bpprc.org). After CDD search, the core region of the two novel insecticidal proteins were identified. The domains I, II, and III of Cry8Ha1 protein were located from amino acids 94 to 290, 300 to 510 and 512 to 657, respectively. While for Cry8Ia1 protein these domain regions were located from amino acids 106 to 288, 296 to 516, and 517 to 665, respectively. BLAST analysis results showed that some domains of the two Cry8H proteins have high similarity to other proteins from the Cry family such as Cry3, Cry1B, or Cry1I, rather than to Cry8 members. Phylogenetic analysis were performed with the complete protoxin or the toxin core sequences of these two Cry8 proteins and compared with different Cry proteins (Figure1). These analyses showed that protoxin sequences of Cry8Ha1 and Cry8Ia1 clustered in a single branch with the other Cry8 protoxins as expected. In contrast the sequence of the toxin cores of these proteins were found in different branches. The toxin core of Cry8Ha1 toxin was found clustered in the same branch with Cry3 proteins together with Cry8Ba, Cry8Pa, Cry8Ca, and Cry8Ja, while Cry8Ia1 was found grouped with Cry8G, Cry8K, and Cry7G toxins (Figure1). Suggesting that these two novel proteins Cry8Ha1 and Cry8Ia1 may have differential specificity. We also performed phylogenetic analyses of the different domains. Analysis of domain I sequences from several Cry proteins showed that this region is distributed in three main clusters (Figure S1). Cluster A includes 11 different Cry8 proteins clustered with domain I sequences from Cry1B and Cry1I (Figure S1, Cluster A); the second group (Figure S1, Cluster B), includes domain I sequences from Cry8Ha1 and Cry8Ia1 that were found to be grouped with other nine Cry8 and Cry3 proteins; the third group (Figure S1, Cluster C) includes six members, forming a cluster including more distant Cry8 proteins (Figure S1). The sequences from domains II and III showed larger variation, where Toxins 2020, 12, 642 3 of 11 the Cry8Ha1 and Cry8Ia1 were located in different clusters. For domain II sequences six different clusters were identified, the Cry8Ha1 was located in Cluster A (Figure S2, Cluster A), which included sequences of domain II from Cry1B and Cry1I proteins, while Cry8Ia1 is found in cluster E with other Cry8 members such as Cry8Ib1, Cry8Ga1, Cry8Ka1, and Cry8Kb1 (Figure S2, Cluster E). Finally, for domain III sequences, they were classified in seven clusters, where the domain III of Cry8Ha1 is found in Cluster A, which includes sequences from Cry1I, Cry1B and Cry3 proteins (Figure S3, Cluster A), while Cry8Ia1 is found in Cluster E together with other Cry8 proteins, such as Cry8Ib1, Cry8Sa1, Cry8Ta,1 and Cry8La1 (Figure S3, Cluster E). These analyses show that Cry8Ha1 toxin is more closely relatedToxins with 2020 Cry3,, 12, x FOR Cry1B, PEER REVIEW and Cry1I. 5 of 11

FigureFigure 1. Phylogenetic 1. Phylogenetic analysis analysis of of the the protoxinprotoxin or or toxi toxinn core core amino amino acid acid sequences sequences from from different diﬀ erentCry Cry proteins.proteins. These These analyses analyses involved involved 52 52 amino amino acid se sequences.quences. Evolutionary Evolutionary analyses analyses were were conducted conducted in in ClustalClustal Omega Omega and and Neighbour-joining Neighbour-joining Phylogenetic Phylogenetic analysis analysis (Madeira (Madeira et al. et al.2019). 2019). The TheCry8Ha1 Cry8Ha1 and and Cry8Ia1 proteins were labeled in red letters. Cry8Ia1 proteins were labeled in red letters.

2.2. DifferentialWe also Toxicity performed of Cry8Ha1 phylogenetic and Cry8Ia1 analyses Protoxins of the anddifferent Activated domains. Toxins Analysis Against of Di ffdomainerent I Coleopteransequences Insects from several Cry proteins showed that this region is distributed in three main clusters (Figure S1). Cluster A includes 11 different Cry8 proteins clustered with domain I sequences from Cry1BTo express and Cry1I the (Figurecry8Ha1 S1,and Clustercry8Ia1 A); thegenes, second primers group (Figure were S1, designed Cluster B), according includes domain to their I gene sequences,sequences amplified from Cry8Ha1 from and the Cry8Ia1 total DNA that were of BtSU4 found strain to be grouped and cloned with other into pSTKnine Cry8 shuttle and Cry3 vector as describedproteins; in the Materials third group and (Figure Methods. S1, Cluster The acrystalliferousC) includes six members, HD-73- formingstrain wasa cluster transformed including with thesemore constructions distant Cry8 by proteins electroporation, (Figure S1). and The the se resultedquences recombinantfrom domains strains II and HD8HIII showed and HD8Ilarger were usedvariation, to express where these the proteinsCry8Ha1 and Cry8Ia1 determine were their located toxicity in different against clusters. different For domain coleopteran II sequences insects as describedsix different below. clusters The morphology were identified, of the thecrystals Cry8Ha1 produced was located by in BtSU4, Cluster HD8H, A (Figure and S2, HD81 Cluster strains A), was which included sequences of domain II from Cry1B and Cry1I proteins, while Cry8Ia1 is found in analyzed in spore/crystal suspensions observed under scanning electron microscopy (SEM). Figure2 cluster E with other Cry8 members such as Cry8Ib1, Cry8Ga1, Cry8Ka1, and Cry8Kb1 (Figure S2, showsCluster that E). BtSU4 Finally, had for typical domain Bt III spores sequences, and they relatively were classified small irregular in seven clusters, crystals where (indicated the domain by arrow). The ICPsIII of Cry8Ha1 accumulated is found in BtSU4 in Cluster crystals A, which were includes composed sequences of 135 from kDa Cry1I, protein, Cry1B as revealed and Cry3 by proteins SDS-PAGE electrophoresis(Figure S3, Cluster (Figure A),2E). while Cry8Ia1 is found in Cluster E together with other Cry8 proteins, such as Cry8Ib1, Cry8Sa1, Cry8Ta,1 and Cry8La1 (Figure S3, Cluster E). These analyses show that Cry8Ha1 toxin is more closely related with Cry3, Cry1B, and Cry1I.

3.2. Differential Toxicity of Cry8Ha1 and Cry8Ia1 Protoxins and Activated Toxins Against Different Coleopteran Insects To express the cry8Ha1 and cry8Ia1 genes, primers were designed according to their gene sequences, amplified from the total DNA of BtSU4 strain and cloned into pSTK shuttle vector as described in Materials and Methods. The acrystalliferous HD-73- strain was transformed with these

ToxinsToxins 20202020,, 1212,, xx FORFOR PEERPEER REVIEWREVIEW 66 ofof 1111 constructionsconstructions byby electroporation,electroporation, andand thethe resultedresulted recorecombinantmbinant strainsstrains HD8HHD8H andand HD8IHD8I werewere usedused toto expressexpress thesethese proteinsproteins andand determinedetermine theirtheir totoxicityxicity againstagainst differentdifferent coleopterancoleopteran insectsinsects asas describeddescribed below.below. TheThe morphologymorphology ofof thethe crystalscrystals producedproduced byby BtSU4,BtSU4, HD8H,HD8H, andand HD81HD81 strainsstrains waswas analyzedanalyzed inin spore/crystalspore/crystal suspensionssuspensions observedobserved underunder scanningscanning electronelectron microscopymicroscopy (SEM).(SEM). FigureFigure 22 showsshows thatthat BtSU4BtSU4 hadhad typicaltypical BtBt sporesspores andand relativerelativelyly smallsmall irregularirregular crystalscrystals (indicated(indicated byby arrow).arrow). TheThe ICPsICPs accumulatedaccumulated inin BtSU4BtSU4 crystalscrystals werewere cocomposedmposed ofof 135135 kDakDa protein,protein, asas revealedrevealed byby SDS-SDS- Toxins 2020, 12, 642 4 of 11 PAGEPAGE electrophoresiselectrophoresis (Figure(Figure 2E).2E).

FigureFigure 2.2. SEMSEMSEM observationobservation observation andand and ElectrophoreticElectrophoretic Electrophoretic analysisanalysis analysis ofof ofBtBt spore Btspore spore andand and crystalcrystal crystal mixtures.mixtures. mixtures. PanelsPanels Panels ((AA–– DD(A),),– DSEMSEM), SEM imagesimages images ofof the ofthe the sporespore spore andand and crystalcrystal crystal mixturesmixtures mixtures ofof of BtBt Bt strainstrain strain BtSU4,BtSU4, BtSU4, HD8H,HD8H, HD8H, HD8IHD8I HD8I andand and crystal crystal negativenegative strainstrain HD-73HD-73 --.-. . TheThe The scalescale scale barbar bar inin in panelspanels panels A,A, A, B,B, B, CC C andand and DD D waswas 2 2 μµμm.m. Panel Panel ( (EE),), Sodium Sodium dodecyl dodecyl sulfate-polyacrylamidesulfate-polyacrylamide gelgel gel electrophoresiselectrophoresis electrophoresis (SDS-PAG(SDS-PAG (SDS-PAGE)E)E) profilesprofiles proﬁles ofof of thesethese these sporespore spore andand and crystalcrystal crystal mixtures.mixtures. mixtures. M,M, Bio-RadBio-Rad high-molecular-masshigh-molecular-mass proteinprotein marker.marker. TheThe otherother laneslanes werewere totaltotal proteinprotein samplessamples fromfrom BtBt strainstrain BtSU4,BtSU4, HD8H,HD8H, HD8IHD8I and and crystal crystal negative negative strain strain HD-73 HD-73--,, respectively.respectively.

TheThe SEMSEM images images ofof Figure Figure 22 2 showed showedshowed thatthatthat bothbothboth Cry8Ha1Cry8Ha1Cry8Ha1 andandand Cry8Ia1Cry8Ia1Cry8Ia1 crystalscrystalscrystals inclusionsinclusionsinclusions accumulatedaccumulated formingforming largelarge irregularirregular crystalscrystals (Figure(Figure 2 2B,C,2B,C,B,C, indicatedindicatedindicated bybyby arrows).arrows).arrows). SDS-PAGESDS-PAGE analysisanalysisanalysis showedshowed thatthat thethe expressedexpressed Cry8Ha1Cry8Ha1 andand Cry8Ia1Cry8Ia1 prpr protoxinsotoxinsotoxins werewere resolvedresolved alsoalso asas 135135 kDa bands bands (Figure(Figure 2 2E),2E),E), similar similarsimilar to toto the thethe ICPs ICPsICPs molecular molecularmolecular weight weightweight band bandband observed observedobserved for forfor BtSU4 BtSU4BtSU4 strain. strain.strain. ToTo compare compare the the toxicity toxicity of of protoxins protoxins with with that that of of activated activated toxinstoxins ofof bothboth Cry8Ha1Cry8Ha1 andand Cry8Ia1Cry8Ia1 proteins,proteins, thethethe protoxins protoxinsprotoxins of ofof Cry8Ha1 Cry8HaCry8Ha and11 andand Cry8Ia1 Cry8Ia1Cry8Ia1 were werewere activated activatedactivated by chymotrypsin byby chymotrypsinchymotrypsin digestion. digestion.digestion. We decided WeWe decideddecidedto activate toto activateactivate Cry8 toxins Cry8Cry8 with toxinstoxins chymotrypsin withwith chymotrypsinchymotrypsin since it sincesince was itit previously waswas previouslypreviously shown shownshown that thisthatthat proteasethisthis proteaseprotease was waswashighly highlyhighly eﬃcient efficientefficient for activation forfor activationactivation of Cry3Aa ofof Cry3AaCry3Aa toxin thattoxintoxin is thatthat toxic isis to toxictoxic coleopteran toto coleopterancoleopteran larvae inlarvaelarvae contrast inin contrastcontrast to trypsin toto trypsinproteasetrypsin proteaseprotease [10]. After [14].[14]. After digestionAfter digestiondigestion with with chymotrypsin,with chymotchymotrypsin,rypsin, the activated thethe activatedactivated toxins toxinstoxins were werewere again againagain analyzed analyzedanalyzed by bybySDS–PAGE SDS–PAGESDS–PAGE electrophoresis, electrophoresis,electrophoresis, showing showingshowing that thatthat the Cry8Ha1thethe Cry8Ha1Cry8Ha1 is activated isis activatedactivated into into ainto 70 a kDaa 7070 kDakDa protein proteinprotein fragment fragmentfragment after afterafter2 h of 22 treatment hh ofof treatmenttreatment with the withwith protease. thethe protease.protease. The 70 The kDaThe 70 protein70 kDakDa fragmentproteinprotein fragmentfragment was relative waswas stable relativerelative since stablestable it remained sincesince itit remainedremainedafter 24 h afterafter of treatment, 2424 hh ofof treatment,treatment, although althoughalthough some of the somesome 70 ofof kDa thethe fragment 7070 kDakDa fragmentfragment was further waswas degraded furtherfurther degradeddegraded into a smaller intointo aa smallersmallerfragment fragmentfragment with a molecular withwith aa molecularmolecular weight about weightweight 55 kDa aboutabout (Figure 5555 kDakDa3). In(Figure(Figure contrast, 3).3). theInIn contrast, protoxincontrast, ofthethe Cry8Ia1 protoxinprotoxin after ofof Cry8Ia1proteaseCry8Ia1 afterafter treatment proteaseprotease showed treatmenttreatment two main showedshowed fragments twotwo mainmain of 97 fragmentsfragments and 66 kDa ofof 9797 (Figure andand 66663). kDakDa (Figure(Figure 3).3).

Figure 3. SDS-PAGE protein proﬁle of Cry8Ha1 and Cry8Ia1 proteins, after digestion with chymotrypsin protease. “M” means the marker, and the molecular weight of the bands are 212, 116, 97, 66 and 45 kDa, respectively.

The toxicity of the two Cry8Ha1 and Cry8Ia1 protoxins was ﬁrst analyzed against diﬀerent insects (Table1, Figure4). The data showed that the Cry8Ha1 protoxin was toxic to both H. parallela larvae and adults. This protoxin was also toxic against H. oblita adults, but not to H. oblita larvae neither to C. bowringi larvae (Table1). Analysis against A. glabripennis larvae showed that Cry8Ha1 protoxin was also toxic to the larvae of this insect (Figure4). The Cry8Ha1 treated larvae showed a severe Toxins 2020, 12, x FOR PEER REVIEW 7 of 11

Figure 3. SDS-PAGE protein profile of Cry8Ha1 and Cry8Ia1 proteins, after digestion with chymotrypsin protease. “M” means the marker, and the molecular weight of the bands are 212, 116, 97, 66 and 45 kDa, respectively.

The toxicity of the two Cry8Ha1 and Cry8Ia1 protoxins was first analyzed against different insects (Table 1, Figure 4). The data showed that the Cry8Ha1 protoxin was toxic to both H. parallela larvae and adults. This protoxin was also toxic against H. oblita adults, but not to H. oblita larvae neither to C. bowringi larvae (Table 1). Analysis against A. glabripennis larvae showed that Cry8Ha1 protoxin was also toxic to the larvae of this insect (Figure 4). The Cry8Ha1 treated larvae showed a severe effect in their growth, they remain small, lack mobility and looks severely damaged, after few Toxinsdays 2020of treatment, 12, 642 they died. The image of Figure 4B shows these effects. In contrast, the Cry8Ia15 of 11 protoxin, showed toxicity only against H. parallela larvae and did not show toxicity to any the other insect tested in this study (Table 1). We then analyzed toxicity of the activated toxin samples, effect in their growth, they remain small, lack mobility and looks severely damaged, after few days of chymotrypsin activation caused the complete loss of toxicity of Cry8Ha1 to H. oblita adults and seems treatment they died. The image of Figure4B shows these e ffects. In contrast, the Cry8Ia1 protoxin, to reduce the activity against H. parallela adults when compared to the Cry8Ha1 protoxin, although showed toxicity only against H. parallela larvae and did not show toxicity to any the other insect these data are not significant since their fiducial limits overlapped. The activation of Cry8Ha1 toxin tested in this study (Table1). We then analyzed toxicity of the activated toxin samples, chymotrypsin with chymotrypsin resulted in enhanced toxicity against C. bowringi larvae (Table 1). The activated activation caused the complete loss of toxicity of Cry8Ha1 to H. oblita adults and seems to reduce the Cry8Ia1 toxin showed no toxicity to any insects tested in this study. activity against H. parallela adults when compared to the Cry8Ha1 protoxin, although these data are not significant since their fiducial limits overlapped. The activation of Cry8Ha1 toxin with chymotrypsin Table 1. The LC50 Cry8Ha1 and Cry8Ia1 against Holotrichia oblita, Holotrichia parallela, and Colaphellus resultedbowringi in enhanced. toxicity against C. bowringi larvae (Table1). The activated Cry8Ia1 toxin showed no toxicity to any insects tested in this study. H. oblita H. parallela C. bowringi Table 1. TheAdult LC Cry8Ha1 and Cry8Ia1 againstAdultHolotrichia oblita, Holotrichia parallela, and Colaphellus 50 Larvae Larvae bowringiLC.50 Values in LC50 Values in Sample Larvae LC50 Values in 108 LC50 Values in mg/mL mg/mL H. oblita8 H. parallela C. bowringi Sample 10 CFU/g CFU/g mg/mL (ConfidenceAdult (ConfidenceAdult Larvae Larvae (Confidence Limits)8 (Confidence Limits) Limits)LC 50 Values in Larvae Limits)LC50 Values in LC50 Values in 10 LC50 Values in mg/mL 108 CFU/g mg/mL CFU/g mg/mL Cry8Ha1-Pro 0.99 (0.62–2.05)(Confidence Limits) NA 0.16 (Confidence(0.00–0.57) Limits) 218.90(Confidence (68.21–661.10) Limits) (Confidence Limits) NA Cry8Ia1-ProCry8Ha1-Pro NA 0.99 (0.62–2.05) NA NA NA 0.16 (0.00–0.57) 85.03 218.90 (30.59–176.08) (68.21–661.10) NA NA Cry8Ha1-ActCry8Ia1-Pro NA NA NT NA 0.66 (0.20–12.36) NA 85.03 (30.59–176.08) NT NA 0.02 (0.01–0.02) Cry8Ha1-Act NA NT 0.66 (0.20–12.36) NT 0.02 (0.01–0.02) Cry8Ia1-ActCry8Ia1-Act NA NA NT NT NA NA NT NT NA NA Note: the the NA NA= no= no activity; activity; NT = NTnot = tested; not tested; “-Pro” =“-Pro”protoxin; = protoxin; “-Act” = activated “-Act” toxin;= activated “CFU” toxin;= the colony-forming “CFU” = the unitcolony-forming of crystal-spore unit mixture. of crystal-spore mixture.

Figure 4. TheThe bioassay bioassay of of Cry8Ha1 Cry8Ha1 protoxin protoxin against against AnoplophoraAnoplophora glabripennis. glabripennis (.(A):A the): the mortality mortality of ofA. A.glabripennis glabripennis treatedtreated by by different different dose dose of of Cry8Ha1 Cry8Ha1 prot protoxin.oxin. This This experimented experimented was performed in triplicate.triplicate. ForFor eacheach repeat, repeat, 24 24 larvae larvae were were tested; tested; (B): Morphology(B): Morphology of toxin-treated of toxin-treatedA. glabripennis A. glabripennislarvae. larvae. 3. Discussion 4. DiscussionThe Cry8 toxin family has been reported to have toxicity against coleopteran insects and until now 60The di Cry8fferent toxin Cry8 family proteins has (including been reported 2 Cry8-like to have proteins) toxicity that against belong coleopteran to 28 holotypes insects by and the until new Bacterialnow 60 different Pesticidal Cry8 Protein proteins Resource (including Center 2 (BPPRC) Cry8-like (https: proteins)//www.bpprc.org that belong)[ to6]. 28 Cry8 holotypes proteins by have the beennew Bacterial shown to Pesticidal be mainly Protein toxic to Scarabs,Resource but Center some (BPPRC) patents claimed (https://www.bpprc.or that certain Cry8g)[6]. proteins Cry8 also proteins show toxicityhave been to Chrysomeloideashown to be mainly insects toxic [11 to]. Scarabs, The larvae but andsome adults patents of bothclaimed Scarabs that andcertain Chrysomeloidea Cry8 proteins insectsalso show feed toxicity on plants, to thusChrysomeloidea it is important insects to find [15]. effective The larvae strategies and to adults control of both both stages Scarabs of these and insects. Until recently, as shown in Figure5, the Cry8Ab, Cry8Ca, Cry8Da, Cry8Db, Cry8Ea, Cry8Ga, Cry8Na, and a Cry8-like protein have been confirmed toxic to six main harmful scarabs. Among these proteins, the Cry8Da, Cry8Db and Cry8Ga proteins have shown to be effective against both adults and larvae stages of the insects [12,13]. In this study, we show that the protoxin from the novel Cry8Ha1 showed toxicity against both adults and larvae of H. parallela. In contrast, this Cry8Ha1 protoxin was only toxic to the adults of H. oblita, but not to the larvae. In addition, Cry8Ha1 was toxic to the larvae of A. glabripennis. The protoxin of the new Cry8Ia1 was only toxic to the larval stages of H. parallela. The reason for the differential toxicities of these protoxins observed in adults or larvae stages of certain species is not known, but one alternative could be related to a differential expression of Cry8 midgut receptors in both developmental stages. This hypothesis remains to be analyzed. Toxins 2020, 12, 642 6 of 11

The phylogenetic analysis of Cry8Ha toxin core sequence showed that this proteins is more related to Cry3 proteins and the analysis of the different domains of Cry8Ha1 and Cry8Ia1 proteins confirmed that Cry8Ha1 is more related to the coleopteran active proteins Cry3, Cry1B, and Cry1I that have been shown to be specifically toxic to Chrysomeloidea insects [5]. Our data suggest that the novel Cry8Ha1 and Cry8Ia1 proteins have evolved from different origins, since these Cry8Ha1 and Cry8Ia1 proteins do not cluster together. Only Domain I of both Cry8Ha1 and Cry8Ia1 proteins clustered together with domain I from Cry3 proteins. Domain II of Cry8Ha1 is more related to domain II from Cry1B and Cry1I proteins; while Domain III of this protein is closer to domain III from the Cry1B, Cry1I, and Cry3 coleopteran active proteins. In contrast Domains II and III of Cry8Ia1 are not related to the corresponding domains of other coleopteran active toxins, only to other Cry8 proteins, such as Cry8Ib and Cry8Ga. Based in these close phylogenetic relationships with other Cry toxins active against Chrysomeloidea insects we decided to determine the toxicity of the two novel Cry8Ha1 and Cry8Ia1 proteins against some Chrysomeloidea pests, such as C. bowringi and A. glabripennis. For C. bowringi, Cry8Ha1 was effective, but the toxicity was revealed only with the Cry8Ha1 activated toxin after in vitro activation. Previously, a similar observation was made with the Cry7Aa1 protein against Colorado potato beetle where only the in vitro activated Cry7Aa1 protein showed to be toxic [9]. It has been shown that the content of midgut proteases may have great variations between different insects orders, for example, the main digestive proteases of Lepidoptera and Diptera are serine proteases, whereas those of Coleoptera are mainly cysteine and aspartic proteases [14]. Therefore, it is possible that the Cry8Ha1 is not processed correctly by the C. bowringi midgut proteases but the in vitro processing could activate the toxin properly revealing its toxicity against C. bowringi larvae. In the case of A. glabripennis, the Cry8Ha1 protoxin was effective against A. glabripennis larvae, which is a Cerambycidae insect that harms many forests plants. Until recently, only Cry3Aa protein was shown to has toxicity to some Cerambycidae species, including Phytoecia rufiventris Gautier, Apriona germari, as well as A. glabripennis [15–17]. The discovering of new insecticidal proteins with different amino acid sequences that may have different modes of action is important to have additional tools for the control of Cerambycidae and Chrysomeloidea insects. The toxicity of Cry8Ha1 to two different Cerambycidae and Chrysomeloidea species suggests that other Cry8 proteins could also potentially show toxicity to different coleopteran species. In addition, we also find that the in vitro solubilization and activation was adverse for Cry8Ha1 activity to scarab adults and also for its toxicity against the larval stages of H. parallela, since a reduction in toxicity was observed with the activated toxin. These data could indicate that the treatment with chymotrypsin inactivates the Cry8Ha1 protoxin or that two different mechanisms exist for protoxin or activated toxin, as previously suggested [15]. The fact that Cry8Ha1 activated toxins increases is toxic effect against C. bowringi larvae indicates that chymotrypsin treatment did not inactivate Cry8Ha1. Rather, the data indicate that both Cry8Ha1 protoxin and activated toxin may have independent toxicity pathways in the different insect species analyzed. In the case of Cry1Ab and Cry1Ac toxins, it has been shown that protoxins and activated toxins exert toxicity by independent pathways, where two different oligomer prepores with different pore formation activity are formed depending if protoxin or activated toxin bind to cadherin receptor [18]. Furthermore, it has been shown that the C-terminal region of Cry1Ab protoxin provides additional binding sites for alkaline phosphatase (ALP) and aminopeptidase N (APN) receptors providing a higher binding affinity of the protoxin to the gut membrane which contributes to the higher toxicity of Cry1Ab protoxin compared to the activated toxin [19]. It remains to identify the different protoxin or activated Cry8Ha1 binding proteins in the different insects analyzed and in the different developmental stages. However, an alternative explanation could be that the differential toxicity observed between Cry8Ha1 protoxin or activated toxin is related to the differential proteases that are present in the different insects analyzed. In any case, our data point out that the differential toxicities of protoxin or activated toxin should be taken into account to decide which form of the toxin should be expressed in transgenic plants, depending on the target pest. Toxins 2020, 12, x FOR PEER REVIEW 9 of 11 has been shown that the C-terminal region of Cry1Ab protoxin provides additional binding sites for alkaline phosphatase (ALP) and aminopeptidase N (APN) receptors providing a higher binding affinity of the protoxin to the gut membrane which contributes to the higher toxicity of Cry1Ab protoxin compared to the activated toxin [23]. It remains to identify the different protoxin or activated Cry8Ha1 binding proteins in the different insects analyzed and in the different developmental stages. However, an alternative explanation could be that the differential toxicity observed between Cry8Ha1 protoxin or activated toxin is related to the differential proteases that are present in the different insects analyzed. In any case, our data point out that the differential toxicities of protoxin or activatedToxins 2020, 12toxin, 642 should be taken into account to decide which form of the toxin should be expressed7 of 11 in transgenic plants, depending on the target pest.

Figure 5. InsecticidalInsecticidal activity activity data data of of Cry8 Cry8 proteins proteins published published before before and and in this in this work work [13,16,24–29]. [12,20–26]. The “A” and “L” labels after the insect name means the “Adult” or “Larvae” stagesstages ofof thosethose insects.insects.

5.4. Conclusions Conclusions In this this paper, paper, we characterized two new insecticidal proteins, Cry8Ha1 and Cry8Ia1. In addition addition to thethe larvicidallarvicidal activity activity against againstH. H. parallela parallela, the, the Cry8Ha1 Cry8Ha1 showed showed to be to toxic be totoxic the adultsto the ofadultsH. parallela of H. parallelaand H. oblita and ,H. and oblita to the, and larvae to the of somelarvae Cerambycidae of some Cerambycidae insects such insects as C. bowringisuch as C.and bowringiA. glabripennis and A.. glabripennisConsidering. theConsidering sequence clusterthe sequence characterization cluster characteri and the insecticidalzation and spectrum the insecticidal of Cry8Ha1, spectrum this study of Cry8Ha1,not only providesthis study new not toolsonly provides to control new coleopteran tools to control insects, coleopteran but alsoa insects, valuable but material also a valuable for the materialongoing for investigation the ongoing of investigation insecticidal of specific insecticidal evolution, specific as evolution, well as potential as well as strategies potential for strategies further fortoxin further improvements. toxin improvements. 5. Materials and Methods Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Figure S1. UPGMA phylogenetic analysis of the amino acid sequences from domain I of Cry proteins, Figure S2. UPGMA 5.1. Bacteria Growth Conditions and Plasmids phylogenetic analysis of the amino acid sequences from domain II of Cry proteins, Figure S3. UPGMA phylogeneticThe BtSU4 trees strainof the amino used inacid this sequ studyences wasfrom isolated domain III from of Cry soil proteins. samples in Hebei province, China. - AuthorHD-73 Contributions:a crystal negative C.S. Btand strain, J.Z. conceived was used and as recipient designed strain the experiments; for the expression C.S., G.Y., of the S.H. novel and Cry8Y.G. performedproteins. Btthe strains experiments; were incubatedM.S., A.B. and for 3L.G. days analyzed in cross-ba the data;ffled J.Z. flasks contributed at 30 ◦C ma withterials shaking and analysis at 230 tools; rpm C.S.,in PB M.S. medium and A.B. (0.5% wrote peptone, the paper. 0.3% All authors beef extract; have read pH and 7.2) agreed for expressing to the published the cry8 versiongenes. of theEscherichia manuscript. coli DH5α strain was used for common transformation, while E. coli SCS110 strain was used to produce Funding: This study was supported by the National Key Research and Development Program of China 2017YFD0201204nonmethylated and plasmid National DNA Natural for Science Bt transformation. Foundation of China The E. No. coli 31872935.strains were grown at 37 ◦C in Luria-Bertani medium (LB: 1% tryptone, 0.5% yeast extract, 1% NaCl; pH 7.0). Plasmids derivative Conflicts of Interest: The authors declared that they have no conflicts of interest to this work. 1 from pSTK [27] were constructed for cloning and expression of the genes. Kanamycin (50 µg mL− )

was added to the media, when appropriate, for selection of E. coli and Bt antibiotic resistant strains.

5.2. Gene Cloning and Sequence Analysis In order to clone ICPs genes from BtSU4 strain, the Roche/454 technology was used to sequence the complete genome of this strain. After genome assembling, performed by Roche 454’s Newbler, the contig00204 (GenBank accession number MT905434) was detected, containing two ICPs genes by using the Basic Local Alignment Search Tool (BLAST, https://ftp.ncbi.nlm.nih.gov/blast/executables/ blast+) and an in-house Bt toxin genes database. The two cry-type genes were deposited in GenBank with the following accession numbers AY897354 and EU381044. The new Cry proteins were annotated by using the Conserved Domains Database (CDD) search (https://www.ncbi.nlm.nih.gov/cdd/). The Phylogenetic analyses of the amino acid sequences were conducted by Neighbour-joining [28] and MEGA X [29]. Toxins 2020, 12, 642 8 of 11

5.3. Protein Expression, Extraction and Activation

Primers were designed to clone the novel ICPs genes. SU4_5 (50-GGA ATT CGA TGA GTC CGA ATA ATC AGA AT-30) and 8H_3 (50-CGC GTC GAC TTA CAT TTC TTC TAC AAT CAA TTC-30) primers were designed to amplify cry8Ha1 gene, while SU4_5 and 8I_3 (50-CTC ATT TCT TCT ACA ATC AAT TCT ACA CTG TC-30) primers were used to amplify cry8Ia1 gene. A KOD DNA polymerase and PTC-100 Peltier Thermal Cycler (MJ Research) was used for PCR, comprising 30 cycles (each cycle was composed of 1 min at 94 ◦C, 1 min at 54 ◦C, and 4 min at 68 ◦C) followed by 10 min incubation at 68 ◦C. The amplified full-length cry8Ha1 and cry8Ia1 genes were inserted into the EcoRI-SalI sites and EcoRI-EcoICRI sites of the pSTK shuttle vector, respectively. The plasmids purified from E. coli DH5α strain were then transformed into E. coli SCS110 for producing nonmethylated recombinant plasmids. Then, the expression vectors containing cry8Ha1 and cry8Ia1 genes were introduced into the crystal negative Bt strain HD-73- by electroporation [27]. The positive transformant colonies were incubated in LB medium supplemented with kanamycin at 30 ◦C until sporulation. The spore/crystal mixtures were collected and washed twice in 1.0 M NaCl and sterile distilled water successively. The final suspension was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 10% acrylamide gel. The crystal/spore mixtures were subsequently solubilized in a pH 10.2 buffer containing 50 mM Na2CO3, 50 mM EDTA, 3% 2-mercaptoethanol, incubated on a rotary shaker at 150 rpm at 0 ◦C for 4 h. The supernatant soluble protoxins were separated by centrifugation at 13,500 g for 10 min and the × protoxins were precipitated by adding 4 M Sodium acetate-Acetic acid (NaAc-HAc) buffer (pH 4.5) until the pH reached 5.0 and then incubated at 4 ◦C for 4 h. The protoxin pellet was collected by centrifugation at 13,500 g and washed with pre-chilled distilled water three times to remove NaAc and × HAc, and then dissolved in 50 mM Na2CO3 (pH 10.2). The protoxins were activated by treatment with chymotrypsin (Sigma) (10:1 w/w). The digestion was carried out in 50 mM Na2CO3 buffer (pH 10.2) at 37 ◦C for 2, 12 or 24 h. Reactions were stopped by chilling in ice. The digested proteins were then boiled 3 min with protein-loading dye and analyzed on a 10% acrylamide SDS–PAGE.

5.4. Scanning Electron Microscopy (SEM) Examination After sporulation in LB medium, Bt spores and crystals were harvested by centrifugation at 13,500 g for 1 min. The pellets were suspended and washed twice in distilled water. SEM micrographs × were taken according to the method described before [27]. The suspension was smear on glass slide and placed on aluminum stubs, dried, and ﬁxed in 1% OsO4. Then, the spore crystal mixtures were sputter-coated with gold in an IB-5 ion coater (EIKO Engineering) for 4 min. The SEM micrographs were taken on a Hitachi S4800 digital scanning microscope (Hitachi High-Tech, Japan) at a voltage of 12 kV

5.5. Insects, Rearing Conditions and Bioassay Adults of H. oblita and H. parallela were collected from Baoding and Cangzhou, Hebei province, China. These adults were reared in plastic boxes filled with 5 cm thick sieved soil and fed with elm (Ulmus pumila L.) leaves, at 25 0.5 C, 16L: 8D photoperiod condition and 60% humidity. The eggs ± ◦ were collected and hatched in the same conditions. For bioassay activity against adults, we used the leaf-dipping method. Before feeding, the cleaned elm leaves were soaked in Cry protoxin protein solutions, and then the surface liquid was dried at room temperature. In each treatment, ten adults were feed with the dipped leaves, and then, we replaced the protein dipped elm leaves and check insect mortality every day. These assays were done in triplicate for each protein concentration, using a two-fold dilution protein concentration series (0.03125 mg/mL, 0.0625 mg/mL, 0.125 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 1.0 mg/mL, and 2.0 mg/mL). For bioassays against larvae, we used a procedure modified from Shu (2009) [20]. Five-day larvae were feed with a mixture of potato with spores/crystal suspension. Firstly, the potato was shred, washed, air dried Toxins 2020, 12, 642 9 of 11 and then mixed with Bt spore/crystal water suspension; then, the mixture was further mixed with sieved soil and divide evenly into four six-well culture plates. In each treatment, 24 larvae were feed separately in each well performed in triplicate and mortality was determined after 14 day. The adults of A. glabripennis were collected from Beijing, China, and reared in plastic boxes filled with pieces of willow branches (Salix babylonica), at 25 0.5 C, 16L: 8D photoperiod condition, 60% ± ◦ humidity. The adults feed on the bark and lay their eggs in the bark. The eggs hatch and larvae bioassay were performed on artificial diet. To prepare the artificial diet, we made willow bark powder and willow xylem powder. Branches with a diameter of 5–6 cm from the growing willow were used to separate the willow bark and xylem. Then, we washed, dried, ground, and sieved (80 meshes) the bark and xylem, respectively. The resulted powders were ready for use. The artificial diet was produced by mixing 60 g willow bark powder, 30 g willow xylem powder, 22.5 g agar, 10 g yeast powder, 1.5 g sorbic acid, 1.5 g methyl 4-hydroxybenzoate, 1.5 g ascorbic acid, and 520 mL sterile water, and sterilized at 121 ◦C for 20 min. To perform bioassays, the Cry proteins were mixed with the artificial diet and divided into a 24-well culture plates, with final protein concentrations of 100 µg/g, and 300 µg/g; then, 24 two-day larvae were feed separately in each well in triplicate in a dark condition, and mortality was determined after 10 days. The C. bowringi Baly population was reared in the lab using rape leaves. The bioassays were performed in triplicate against larvae by using the leaf dipping method. Before feeding, the cleaned rape leaves were soaked in Cry protein solutions with two-fold dilution series of protein concentrations of 0.125 µg/mL, 0.0625 µg/mL, 0.03125 µg/mL, 0.01563 µg/mL, and 0.007815µg/mL, and then the surface liquid was dried at room temperature. In each treatment, 20 larvae were feed with the dipped leaves, at 25 0.5 C, 16L: 8D photoperiod, 60% humidity, the mortality was determined after two days. ± ◦ As mentioned above three replicates were performed for each treatment for all bioassays. The 50% lethal concentrations (LC50) were determined by Probit analysis. For all leaf dipping method, 0.005% Triton X-100 was added to increase fluid ductility and allowing uniform protein dip.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6651/12/10/642/s1. Figure S1. UPGMA phylogenetic analysis of the amino acid sequences from domain I of Cry proteins, Figure S2. UPGMA phylogenetic analysis of the amino acid sequences from domain II of Cry proteins, Figure S3. UPGMA phylogenetic trees of the amino acid sequences from domain III of Cry proteins. Author Contributions: C.S. and J.Z. conceived and designed the experiments; C.S., G.Y., S.H. and Y.G. performed the experiments; M.S., A.B. and L.G. analyzed the data; J.Z. contributed materials and analysis tools; C.S., M.S. and A.B. wrote the paper. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the National Key Research and Development Program of China 2017YFD0201204 and National Natural Science Foundation of China No. 31872935. Conﬂicts of Interest: The authors declared that they have no conﬂicts of interest to this work.

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